Isolation and Identification of a Plasmatocyte- spreading Peptide from the Hemolymph of the Lepidopteran Insect. Pseudoplusia. includens. Abstract. Insect blood cells (hemocytes) play an essential role in defense against parasites and other pathogenic organisms that infect. A key class of hemocytes involved in insect cellular immunity is plasmatocytes. Here we describe the isolation and. Pseudoplusia includens that mediates the spreading of plasmatocytes to foreign surfaces. This peptide, designated plasmatocyte- spreading peptide. PSP1), contains 2. H- ENFNGGCLAGYMRTADGRCKPTF- OH. In vitro assays using the synthetic peptide at concentrations . Injection of this peptide into P. Labeling studies indicated that this peptide induced. G3. A3 and 4. 3E9. A8 to spread, whereas plasma induced. This suggests that only a certain subpopulation of plasmatocytes responds to the. Elimination of organisms. Large metazoan parasites are usually killed by encapsulation, a process in which certain classes of hemocytes recognize. In contrast, smaller. In insects like moths and butterflies (Lepidoptera), granular cells and plasmatocytes are the primary classes of hemocytes. However, little is known about the molecules mediating the function and trafficking of these cells (3, 8). The activity of hemocytes is affected in some insect species by endocrine factors, eicosanoids, and biogenic amines (9- 1. Epidermal tissue, fat body, and hemolymph also contain unidentified peptidyl factors that affect hemocyte function or the. While these and other factors from invertebrates have the biochemical and functional properties of vertebrate cytokines. Peptide PSP1 (plasmatocyte-spreading peptide 1). Its precursor protein. Protein-Dependent Transactivation Fei Wu, Rui Xu. Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities. First, the small size of most. Second, in vitro bioassays are required to characterize the response of hemocytes to specific molecules, yet insect blood cells are notoriously. In our studies of the moth Pseudoplusia includens, we have developed assay methods that allow us to manipulate hemocytes in vitro for extended periods of time (1. Using these methods, we observed that cell- free hemolymph (plasma) from P. Herein we report the purification and sequence of a 2. P. Moths were fed 2. Briefly, 3. 6–4. 8- h fifth instar larvae were anesthetized with CO2 and bled from a cut across the last abdominal segment directly into anticoagulant solution (9. Na. OH, 1. 86 mm Na. Cl, 1. 7 mm Na. 2EDTA, and 4. H 4. 5) (2. 2). After a 3. Plasmatocytes and granular cells account for . The other nonadhesive classes of hemocytes in P. Into each well was. Hemocytes were monitored by phase- contrast microscopy using a Nikon TMS inverted microscope. Due to the limited. HPLC1 fractions, bioassays with these fractions were carried out in a 5- . Each experiment consisted of placing 1. The final hemocyte density was 1 . Hemocytes were monitored using a Nikon Diaphot. Hoffman modulation- contrast optics. Bioassays using the synthetic peptide were conducted in 9. The proportion of plasmatocytes spread in each assay was scored at specific periods by. Plasmatocytes were scored as spread if they developed a flattened. When bioassaying chromatography fractions, we considered a region of a chromatogram as containing an active peak if material. The constitutively expressed vanillic acid-sensitive olfactory G protein. A closed-loop synthetic gene circuit for the treatment of diet-induced obesity. A novel G 1-specific enhancer identified in the human heat shock protein 70. Liquid fructose in Western-diet-fed mice impairs liver insulin. Larvae injected with PBS only served as. Larvae were anesthetized with carbon dioxide and injected through a proleg using a glass needle mounted on a micromanipulator. After 1 h, hemocytes were fixed and processed for. Two- hundred cells from a randomly selected field of view were counted. Briefly, hemocytes were fixed in 1. Formalin for 1. 0 min, rinsed with PBS, and permeabilized for 1. PBS plus 0. 1%. Triton X- 1. PBT). Cells were blocked for 1 h in 3% bovine serum albumin (fraction V, Boehringer Mannheim) in PBT, followed. After rinsing four times in PBT, hemocytes were incubated with fluorescein. Texas Red- conjugated goat anti- mouse secondary antibody (Ig. G; Jackson Immuno. Research Laboratories, Inc.). Samples were examined, and the number of labeled hemocytes was counted using the Nikon Diaphot microscope. Immediately after the hemolymph was collected, glutathione (1. H2. O) was added to a final concentration of 5 mm to inhibit melanization (1. Hemocytes were pelleted in a benchtop centrifuge (Eppendorf 5. C) for 5 min at 2. The resulting cell- free hemolymph, referred to as plasma, was stored at . Boiled plasma was also treated with carboxypeptidase Y. Pierce), aminopeptidase M (Pierce), trypsin (Sigma), or proteinase K (Life Technologies, Inc.) according to the manufacturer’s. Da filter. Other samples of plasma were boiled for 4 min in 1. Eppendorf centrifuge. Hints for regulation mechanisms of carotenogenesis in Duanliella bardawil.The supernatant was then passed through a 1. Da cutoff filter. Three- ml fractions were eluted with 1 mm MOPS (Fisher), p. H 7. 2. Strong anion- exchange chromatography was performed in a Kontes 3. One- ml fractions were eluted from the column using 0. Na. MOPS, p. H 7. Active fractions were pooled, lyophilized, and resuspended in HPLC- grade H2. O at a concentration 1. Further purification was achieved by HPLC using a Rheodyne 9. Hitachi L- 6. 22. Hitachi L- 4. 50. A photodiode array detector, and Hitachi D- 7. Separations. were performed on a 5- . Peaks were. eluted with HPLC- grade H2. O using a linear gradient of 0–4. Both H2. O and acetonitrile contained 0. Active fractions from three such HPLC runs (combined total of 1. After. resuspension in 4. Final purification was achieved by rechromatographing on the same HPLC column using a shallower. Fractions were lyophilized overnight, resuspended. The biologically active peak was dried in a Speed- Vac, yielding 1. This represents a 5. Our determination. PSP1- specific antibody,2 which indicates that the concentration of PSP1 in wandering stage (fifth instar)P. Amino acid composition analysis. HCl and 0. 0. 2% . Identity of cysteine residues was determined by performic acid oxidation. The peptide was synthesized in the. Milli. Gen 9. 05. Fmoc chemistry and Fmoc- Phe- Nova. Syn KA1. 00 resin. The peptide. was released from the resin, and the protecting groups were removed with standard King’s reagent. To ensure that the desired. Mass spectral data were generated on a Bruker REFLEX II mass spectrometer operating in a reflection and. The matrix was 2,5- dihydroxybenzoic acid. Sequence analysis was performed using the Genetics Computer Group package (2. When plasma was fractionated using a 1. Da cutoff filter, we found that > 8. Da fraction, whereas almost no plasmatocytes spread when cultured in Ex- cell 4. Fig. 1). Serial dilution of the < 1. Da fraction resulted in a linear decrease in the proportion of spread plasmatocytes, indicating. Similar results were obtained using the flow- through. Da Centricon cutoff filter (data not shown). A, effect of plasma concentration (v/v) on spreading of plasmatocytes in vitro. Spreading was assayed by observing cells after 1 h in culture with plasma. Each data point is the mean percentage . C, micrograph of spread (S) plasmatocytes 1 h after culture in medium supplemented with 8. P. D, micrograph of a hemocyte aggregation (A) formed 1 h after culture in the > 1. Da fraction from boiled plasma (see “Results”). Few plasmatocytes. However, this material. Fig. The boiled plasma was then passed through a 1. Da cutoff filter to estimate the size of the aggregating factor(s). Incubation. of hemocytes in the > 1. Da fraction induced hemocytes to aggregate, whereas the < 1. Da flow- through induced > 7. No further characterization of the factor(s) in the > 1. Da. fraction was pursued during this study. However, because boiling removed most of the protein from plasma, we used this procedure. As a final preliminary experiment, we treated boiled plasma. K, aminopeptidase M, and carboxypeptidase Y and then passed this material through a < 1. Da filter. Based on these results, we concluded that the spreading. N or C terminus. Because. Spreading activity usually eluted as a single peak (fractions 4. This earlier eluting peak was not included in the subsequent. Bioassays using fractions 4. Da cutoff filter. Sixty drop fractions were collected (open circles) and bioassayed for the ability to induce spreading of plasmatocytes. B, strong anion- exchange chromatography of pooled active fractions from the gel filtration column. Fractions 4. 1–5. The sample was then loaded onto a Bio- Rad anion- exchange AG 1- X2 resin column and monitored at. A2. 10. The columns were run sequentially. Horizontal bars indicate fractions that induced > 5. Spreading of plasmatocytes in response. Fig. Active material from the gel filtration column. Na. MOPS, p. H 7. Monitoring at A2. Fig. A minor amount of spreading activity appeared within the first major peak (fraction 1. A2. 10 peak (fractions 1. While two separate active peaks may have been present, they could not be completely resolved, so. These results also suggested that the active compound(s) must be only weakly negatively. Active fractions from the strong anion- exchange column. HPLC (Fig. 3. A). The region from 5. These fractions were therefore rechromatographed on the same HPLC column using a shallower acetonitrile gradient. Fig. Although not completely base line- resolved, the spreading activity from this region was associated predominantly with a. This peak was collected and lyophilized for further analysis. Fractions 1. 4–2. The sample was then loaded onto a 5- . Peaks were eluted with HPLC- grade H2. O using a linear acetonitrile gradient (0–8. A2. 10. The flow rate was 1 ml/min. Fractions were collected manually into glass tubes. The pooled active fractions from three such. HPLC runs (1. 1 ml total of the injected volume) were lyophilized overnight and then resuspended in 4. Spreading of plasmatocytes in response to each fraction was assayed as described under “Experimental Procedures.”B, C1. HC reverse- phase pooled active fractions (4. The flow rate was 1 ml/min. Fractions were lyophilized overnight and resuspended in. Horizontal bars indicate fractions that induced > 5. Spreading of plasmatocytes in response. While 2 internal amino acids (residues 7 and 1. We repeated our isolation of this HPLC peak.
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